Combination therapies for B-cell lynphomas comprising administration of anti-CD20 antibody

ABSTRACT

New combined therapeutic regimens for treatment of B-cell lymphomas are disclosed which comprise in particular administration of anti-CD20 antibodies to patients having low-, intermediate- or high-grade non-Hodgkins lymphomas.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application take priority from Provisional Application No.60/096,180, filed on Aug. 11, 1998; the entire content of which ishereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The invention relates to the use of anti-CD20 antibodies orfragments thereof in the treatment of B-cell lymphomas, particularly theuse of such antibodies and fragments in combined therapeutic regimens.

BACKGROUND OF THE INVENTION

[0003] The use of antibodies to the CD20 antigen as diagnostic and/ortherapeutic agents for B-cell lymphoma has previously been reported.CD20 is a useful marker or target for B-cell lymphomas as this antigenis expressed at very high densities on the surface of malignant B-cells,i.e., B-cells wherein unabated proliferation can lead to B-celllymphomas.

[0004] CD20 or Bp35 is a B-lymphocyte-restricted differentiation antigenthat is expressed during early pre-B-cell development and remains untilplasma cell differentiation. It is believed by some that the CD20molecule may regulate a step in the B-cell activation process which isrequired for cell cycle initiation and differentiation. Moreover, asnoted, CD20 is usually expressed at very high levels on neoplastic(“tumor”) B-cells. The CD20 antigen is appealing for targeted therapy,because it does not shed, modulate, or internalize.

[0005] Previous reported therapies involving anti-CD20 antibodies haveinvolved the administration of a therapeutic anti-CD20 antibody eitheralone or in conjunction with a second radiolabeled anti-CD20 antibody,or a chemotherapeutic agent.

[0006] In fact the Food and Drug Administration has approved thetherapeutic use of one such anti-CD20 antibody, Rituxan®, for use inrelapsed and previously treated low-grade non-Hodgkin's lymphoma (NHL).Also, the use of Rituxan® in combination with a radiolabeled murineanti-CD20 antibody has been suggested for the treatment of B-celllymphoma.

[0007] However, while anti-CD20 antibodies and, in particular, Rituxan®(U.S.; in Britain, MabThera®; in general Rituximab®), have been reportedto be effective for treatment of B-cell lymphomas, such as non-Hodgkin'slymphoma, the treated patients are often subject to disease relapse.Therefore, it would be beneficial if more effective treatment regimenscould be developed. More specifically, it would be advantageous ifanti-CD20 antibodies had a beneficial effect in combination with otherlymphoma treatments, and if new combined therapeutic regimens could bedeveloped to lessen the likelihood or frequency of relapse. Also, itwould be helpful if current treatment protocols for B-cell lymphoma wereimproved whereby patients with lymphomas which are refractory to othertreatment methods could be treated with chimeric or radiolabeledanti-CD20 antibodies. It would also be helpful if treatment withanti-CD20 antibodies, particularly in combination with other treatments,could be used as therapy for other types of lymphoma besides low grade,follicular non-Hodgkins lymphoma (NHL).

SUMMARY OF THE INVENTION

[0008] The present invention discloses combined therapeutic treatmentsfor B-cell lymphomas, and reports the benefits of treating relapsed orrefractory B-cell lymphomas with chimeric and radiolabeled anti-CD20antibodies. In particular, it has been found that treatment withanti-CD20 antibody provides a beneficial synergistic effect whenadministered in combination with cytokines, radiotherapy, myeloablativetherapy, or chemotherapy. Surprisingly, patients who had prior bonemarrow or stem cell transplantation had an unexpected increase in theover-all response rate when compared with patients with no priortherapy.

DETAILED DESCRIPTION OF THE INVENTION

[0009] This invention encompasses combined therapeutic regimens for thetreatment of B-cell lymphomas. In general, such methods include a methodfor treating relapsed B-cell lymphoma, where a patient having priortreatment for lymphoma has relapsed and is administered atherapeutically effective amount of a chimeric anti-CD20 antibody. Suchprior treatments can include, for example, previous treatment withanti-CD20 antibodies, treatments which included a bone marrow or stemcell transplantation, radiotherapy and chemotherapy. The previouschemotherapy may be selected from a wide group of chemotherapeuticagents and combination regimens, including CHOP, ICE, Mitozantrone,Cytarabine, DVP, ATRA, Idarubicin, hoelzer chemotherapy regime, La Lachemotherapy regime, ABVD, CEOP, 2-CdA, FLAG & IDA with or withoutsubsequent G-CSF treatment), VAD, M & P, C-Weekly, ABCM, MOPP and DHAP.

[0010] Also included in the methods of the invention are methods fortreating a subject having B-cell lymphoma wherein the subject isrefractory for other therapeutic treatments, including all those listedabove, i.e., treatment with chimeric anti-CD20 antibody, treatmentswhich included a bone marrow or stem cell transplantation, radiotherapyand chemotherapy. In particular, encompassed are methods of treating apatient who has not exhibited appreciable tumor remission or regressionafter administration of a chimeric anti-CD20 antibody, comprisingadministering to said patient a radiolabeled anti-CD20 antibody.

[0011] In particular, the methods of treating a patient with aradiolabeled antibody after a chimeric antibody are performed wherebythe radiolabeled anti-CD20 antibody is administered from about one weekto about two years after said administration of said chimeric anti-CD20antibody. More particularly, the radiolabeled anti-CD20 antibody isadministered from about one week to about nine months after saidadministration of said chimeric anti-CD20 antibody.

[0012] While any anti-CD20 antibodies can be used for the methods of thepresent invention, a preferred chimeric antibody is C2B8 (IDECPharmaceuticals, Rituximab®). A preferred radiolabeled antibody is Y2B8,which is a murine antibody labeled with yttrium-90 (⁹⁰Y). However,antibodies with other radiolabels may be used, particularly thoselabeled with a beta or alpha isotope. Anti-CD19 antibodies may also beused.

[0013] One of skill in the art would know the parameters for choosing aparticular type of anti-CD20 antibody. For instance, chimeric andhumanized antibodies are beneficial for decreased immunogenicity, andfor facilitating antibody effector mediated immune reactions via thehuman constant region domains. Murine and other mammalian antibodies, incontrast, are beneficial for delivering a radiolabel to the tumor cell,as such antibodies generally have a decreased half-life in vivo.

[0014] Antibody treatments performed initially to which patients arerefractory or have relapsed may include initial treatments with chimericantibodies or mammalian antibodies. Also encompassed are initialtreatments with other antibodies, including anti-CD19 antibodies andanti-Lym antibodies, and treatments with antibodies labeled withcytotoxic moieties, such as toxins, and radiolabels, e.g., Oncolym®(Techniclone) or Bexxar (Coulter).

[0015] It should be clear that the combined therapeutic regimens of thepresent invention can be performed whereby said therapies are givensimultaneously, i.e., the anti-CD20 antibody is administeredconcurrently or within the same time frame (i.e., the therapies aregoing on concurrently, but the agents are not administered precisely atthe same time). The anti-CD20 antibodies of the present invention mayalso be administered prior to or subsequent to the other therapies.Sequential administration may be performed regardless of whether thepatient responds to the first therapy to decrease the possibility ofremission or relapse.

[0016] The combined therapies of the present invention include a methodfor treating B-cell lymphoma comprising administering at least onechimeric anti-CD20 antibody and at least one cytokine. In particular,the invention includes a method for treating B-cell lymphoma comprisingadministering a synergistic therapeutic combination comprising at leastone anti-CD20 antibody and at least one cytokine, wherein thetherapeutic effect is better than the additive effects of either therapyadministered alone. Preferred cytokines are selected from the groupconsisting of alpha interferon, gamma interferon, IL-2, GM-CSF andG-CSF. Again, the anti-CD20 antibody and the cytokine(s) may beadministered sequentially, in either order, or in combination.

[0017] Also included in the present invention is a method for treatingB-cell lymphoma comprising administering to a patient a therapeuticallyeffective amount of a chimeric anti-CD20 antibody before, during orsubsequent to a chemotherapeutic regimen. Such a chemotherapy regimenmay be selected from the group consisting of, at the very least, CHOP,ICE, Mitozantrone, Cytarabine, DVP, ATRA, Idarubicin, hoelzerchemotherapy regime, La La chemotherapy regime, ABVD, CEOP, 2-CdA, FLAG& IDA with or without subsequent G-CSF treatment), VAD, M & P, C-Weekly,ABCM, MOPP and DHAP.

[0018] Also encompassed are methods for treating B-cell lymphomacomprising administering to a patient a therapeutically effective amountof a chimeric anti-CD20 antibody before, during or subsequent to a bonemarrow or peripheral stem cell transplant. Such bone marrow transplantmay also be accompanied by other therapeutic regimens such aschemotherapy. The antibodies of the present invention may also be usedin a method of reducing residual CD20+ tumor cells in bone marrow orstem cells before or after myeloablative therapy by administering to apatient a chimeric anti-CD20 antibody. It may also be possible to usesuch antibodies in vitro to induce apoptosis of tumor cells and reduceor cure bone marrow or stem cell preparations of residual tumor cellsbefore they are infused back into the patient.

[0019] It should be understood that stem cell transplants may beallogeneic or autologous. If the transplant is allogeneic, i.e., fromanother person, the disclosed therapeutic regimens may includetreatments with immunosuppressive drugs before administration of theanti-CD20 antibodies. Coadministration of other drugs designed toenhance acceptance of the transplant and stimulate the production anddifferentiation of immune cells is also contemplated. For instance, ithas been shown that administration of GM-CSF to marrow transplantrecipients promotes the development of specific bone marrow cells whichin turn produces circulating infection-fighting neutrophils, andincreased the survival rate of marrow transplant recipients.

[0020] The methods of the present invention may be used to treat avariety of B-cell lymphomas, including low grade/follicularnon-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediategrade/follicular NHL, intermediate grade diffuse NHL, high gradeimmunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL, bulky disease NHL and Waldenstrom'sMacroglobulinemia. It should be clear to those of skill in the art thatthese lymphomas will often have different names due to changing systemsof classification, and that patients having lymphomas classified underdifferent names may also benefit from the combined therapeutic regimensof the present invention.

[0021] For instance, a recent classification system proposed by Europeanand American pathologists is called the Revised European AmericanLymphoma (REAL) Classification. This classification system recognizesMantle cell lymphoma and Marginal cell lymphoma among other peripheralB-cell neoplasms, and separates some classifications into grades basedon cytology, i.e., small cell, mixed small and large, large cell. Itwill be understood that all such classified lymphomas may benefit fromthe combined therapies of the present invention.

[0022] The U.S. National Cancer Institute (NCI) has in turn divided someof the REAL classes into more clinically useful “indolent” or“aggressive” lymphoma designations. Indolent lymphomas includefollicular cell lymphomas, separated into cytology “grades,” diffusesmall lymphocytic lymphoma/chronic lymphocytic leukemia (CLL),lymphoplasmacytoid/Waldenstrom's Macroglobulinemia, Marginal zonelymphoma and Hairy cell leukemia. Aggressive lymphomas include diffusemixed and large cell lymphoma, Burkitt's lymphoma/diffuse smallnon-cleaved cell lymphoma, Lymphoblastic lymphoma, Mantle cell lymphomaand AIDS-related lymphoma. These lymphomas may also benefit from thecombined therapeutic regimens of the present invention.

[0023] Non-Hodgkin's lymphoma has also been classified on the basis of“grade” based on other disease characteristics including low-grade,intermediate-grade and high-grade lymphomas. Low-grade lymphoma usuallypresents as a nodal disease, and is often indolent or slow-growing.Intermediate- and high-grade disease usually presents as a much moreaggressive disease with large extranodal bulky tumors. Intermediate- andhigh-grade disease, as well as low grade NHL, may benefit from thecombined therapeutic regimens of the present invention.

[0024] The Ann Arbor classification system is also commonly used forpatients with NHL. In this system, stages I, II, III, and IV of adultNHL can be classified into A and B categories depending on whether thepatient has well-defined generalized symptoms (B) or not (A). The Bdesignation is given to patients with the following symptoms:unexplained loss of more than 10% body weight in the 6 months prior todiagnosis, unexplained fever with temperatures above 38° C. anddrenching night sweats. Occasionally, specialized staging systems areused:

[0025] Stage I—involvement of a single lymph node region or localizedinvolvement of a single extralymphatic organ or site.

[0026] Stage II—involvement of two or more lymph node regions on thesame side of the diaphragm or localized involvement of a singleassociated extralymphatic organ or site and its regional lymph nodeswith or without other lymph node regions on the same side of thediaphragm.

[0027] Stage III—involvement of lymph node regions on both sides of thediaphragm, possibly accompanying localized involvement of anextralymphatic organ or site, involvement of the spleen, or both.

[0028] Stage IV—disseminated (multifocal) involvement of 1 or moreextralymphatic sites with or without associated lymph node involvementor isolated extralymphatic organ involvement with distant (non-regional)nodal involvement.

[0029] For further details, see The International Non-Hodgkin's LymphomaPrognostic Factors Project: A predictive model for aggressivenon-Hodgkin's lymphoma. New England J. Med. 329(14): 987-994 (1993).

[0030] Preferred antibodies, dosage regimens and particular combinationsof therapy will now be illustrated by way of the following exemplarydata.

[0031] Rituximab® and Y2B8

[0032] Non-Hodgkin's lymphoma (NHL) affects approximately 250,000 peoplein the United States. The majority of patients with NHL are not cured bychemotherapy, radiotherapy, or high-dose treatment with autologous bonemarrow (ABMT) or peripheral blood stem cell (PBSC) support.

[0033] Approximately 80% of non-Hodgkin's lymphomas are B-cellmalignancies and >95% of these express the CD20 antigen on the cellsurface. This antigen is an attractive target for immunotherapy becauseit is found exclusively on B-cells, and not on hematopoietic stem cells,pro-B-cells, normal plasma cells, or other normal tissues. It is notshed from the cell surface and does not modulate upon antibody binding(1).

[0034] Rituximab® is one of a new generation of monoclonal antibodiesdeveloped to overcome limitations encountered with murine antibodies,including short half-life, limited ability to stimulate human effectorfunctions, and immunogenicity (2,3).

[0035] Rituximab® is a genetically engineered monoclonal antibody withmurine light- and heavy-chain variable regions and human gamma Iheavy-chain and kappa light-chain constant regions. The chimericantibody is composed of two heavy chains of 451 amino acids and twolight chains of 213 amino acids and has an approximate molecular weightof 145 kD. Rituximab® is more effective than its murine parent in fixingcomplement and mediating ADCC, and it mediates CDC in the presence ofhuman complement (4). The antibody inhibits cell growth in the B-celllines FL-18, Ramos, and Raji, sensitizes chemoresistant human lymphomacell lines to diphtheria toxin, ricin, CDDP, doxorubicin, and etoposide,and induces apoptosis in the DHL-4 human B-cell lymphoma line in adose-dependent manner (5). In humans, the half-life of the antibody isapproximately 60 hours after the first infusion and increases with eachdose to 174 hours after the fourth infusion. The immunogenicity of theantibody is low; of 355 patients in seven clinical studies, only three(<1%) had a detectable anti-chimeric antibody (HACA) response.

[0036] Rituximab® was genetically engineered using the murine 2B8antibody. The 2B8 antibody has also been conjugated to differentradiolabels for diagnostic and therapeutic purposes. To this end,copending application Ser. Nos. 08/475,813, 08/475,815 and 08/478,967,herein incorporated by reference in their entirety, discloseradiolabeled anti-CD20 conjugates for diagnostic “imaging” of B-celllymphoma tumors before administration of therapeutic antibody. “In2B8”conjugate comprises a murine monoclonal antibody, 2B8, specific to humanCD20 antigen, that is attached to Indium[111] (¹¹¹In) via a bifunctionalchelator, i.e., MX-DTPA (diethylene-triaminepentaacetic acid), whichcomprises a 1:1 mixture of 1-isothiocyanatobenzyl-3-methyl-DTPA and1-methyl-3-isothiocyanatobenzyl-DTPA. Indium-[111] is selected as adiagnostic radionuclide because it emits gamma radiation and finds priorusage as an imaging agent.

[0037] Patents relating to chelators and chelator conjugates are knownin the art. For instance, U.S. Pat. No. 4,831,175 of Gansow is directedto polysubstituted diethylenetriaminepentaacetic acid chelates andprotein conjugates containing the same, and methods for theirpreparation. U.S. Pat. Nos. 5,099,069, 5,246,692, 5,286,850, and5,124,471 of Gansow also relate to polysubstituted DTPA chelates. Thesepatents are incorporated herein in their entirety.

[0038] The specific bifunctional chelator used to facilitate chelationin application Ser. Nos. 08/475,813, 08/475,815 and 08/478,967 wasselected as it possesses high affinity for trivalent metals, andprovides for increased tumor-to-non-tumor ratios, decreased bone uptake,and greater in vivo retention of radionuclide at target sites, i.e.,B-cell lymphoma tumor sites. However, other bifunctional chelators areknown in the art and may also be beneficial in tumor therapy.

[0039] Also disclosed in application Ser. Nos. 08/475,813, 08/475,815and 08/478,967 are radiolabeled therapeutic antibodies for the targetingand destruction of B-cell lymphomas and tumor cells. In particular, theY2B8 conjugate comprises the same anti-human CD20 murine monoclonalantibody, 2B8, attached to yttrium-[90] (⁹⁰Y) via the same bifunctionalchelator. This radionuclide was selected for therapy for severalreasons. The 64 hour half-life of ⁹⁰Y is long enough to allow antibodyaccumulation by the tumor and, unlike e.g. ¹³¹i, it is a pure betaemitter of high energy with no accompanying gamma irradiation in itsdecay, with a range of 100 to 1000 cell diameters. The minimal amount ofpenetrating radiation allows for outpatient administration of⁹⁰Y-labeled antibodies. Furthermore, internalization of labeledantibodies is not required for cell killing, and the local emission ofionizing radiation should be lethal for adjacent tumor cells lacking thetarget antigen.

[0040] Because the ⁹⁰Y radionuclide was attached to the 2B8 antibodyusing the same bifunctional chelator molecule MX-DTPA, the Y2B8conjugate possesses the same advantages discussed above, e.g., increasedretention of radionuclide at a target site (tumor). However, unlike¹¹¹In, it cannot be used for imaging purposes due to the lack of gammaradiation associated therewith. Thus, a diagnostic “imaging”radionuclide, such as ¹¹¹In, can be used for determining the locationand relative size of a tumor prior to and/or following administration oftherapeutic chimeric or ⁹⁰Y-labeled antibodies in the combined regimensof the invention. Additionally, indium-labeled antibody enablesdosimetric assessment to be made.

[0041] Depending on the intended use of the antibody, i.e., as adiagnostic or therapeutic reagent, other radiolabels are known in theart and have been used for similar purposes. For instance, radionuclideswhich have been used in clinical diagnosis include ¹³¹I, ¹²⁵I, ¹²³I,⁹⁹Tc, ⁶⁷Ga, as well as ¹¹¹In. Antibodies have also been labeled with avariety of radionuclides for potential use in targeted immunotherapy(Peirersz et al. (1987) The use of monoclonal antibody conjugates forthe diagnosis and treatment of cancer. Immunol. Cell Biol. 65: 111-125).These radionuclides include ¹⁸⁸Re and ¹⁸⁶Re as well as ⁹⁰Y, and to alesser extent ¹⁹⁹Au and ⁶⁷Cu. I-(131) has also been used for therapeuticpurposes. U.S. Pat. No. 5,460,785 provides a listing of suchradioisotopes and is herein incorporated by reference.

[0042] As reported in copending application Ser. Nos. 08/475,813,08/475,815 and 08/478,967, administration of the radiolabeled Y2B8conjugate, as well as unlabeled chimeric anti-CD20 antibody, resulted insignificant tumor reduction in mice harboring a B-cell lymphoblastictumor. Moreover, human clinical trials reported therein showedsignificant B-cell depletion in lymphoma patients infused with chimericanti-CD20 antibody. In fact, chimeric 2B8 has recently been heralded thenation's first FDA-approved anti-cancer monoclonal antibody under thename of Rituxan®. Thus, at least one chimeric anti-CD20 antibody hasbeen shown to demonstrate therapeutic efficacy in the treatment ofB-cell lymphoma.

[0043] In addition, U.S. application Ser. No. 08/475,813, hereinincorporated by reference, discloses sequential administration ofRituxan®, a chimeric anti-CD20, with both or either indium-labeled oryttrium-labeled murine monoclonal antibody. Although the radiolabeledantibodies used in these combined therapies are murine antibodies,initial treatment with chimeric anti-CD20 sufficiently depletes theB-cell population such that the HAMA response is decreased, therebyfacilitating a combined therapeutic and diagnostic regimen.

[0044] Thus, in this context of combined immunotherapy, murineantibodies may find particular utility as diagnostic reagents. Moreover,it was shown in U.S. application Ser. No. 08/475,813 that atherapeutically effective dosage of the yttrium-labeled anti-CD20antibody following administration of Rituxan® is sufficient to (a) clearany remaining peripheral blood B-cells not cleared by the chimericanti-CD20 antibody; (b) begin B-cell depletion from lymph nodes; or (c)begin B-cell depletion from other tissues.

[0045] Thus, conjugation of radiolabels to cancer therapeutic antibodiesprovides a valuable clinical tool which may be used to assess thepotential therapeutic efficacy of such antibodies, create diagnosticreagents to monitor the progress of treatment, and devise additionaltherapeutic reagents which may be used to enhance the initialtumor-killing potential of the chimeric antibody. Given the provenefficacy of an anti-CD20 antibody in the treatment of non-Hodgkin'slymphoma, and the known sensitivity of lymphocytes to radioactivity, itwould be highly advantageous for such chimeric and radiolabeledtherapeutic antibodies to find use in combined therapeutic regimenswhich decrease the frequency of relapsed or refractory non-Hodgkin'slymphoma. In addition, it would be beneficial if such combinedtherapeutic regimens found use in the treatment of other B-celllymphomas.

[0046] Low-Grade ir Follicular NHL

[0047] Single-Agent Studies with Relapsed or Refractory NHL

[0048] FDA approval of Rituxinmab® was based on five single-agentstudies primarily in patients with low-grade or follicular NHL. An earlyPhase I study of single Rituximab® infusions ranging from 10-500 mg/m²demonstrated that the maximum tolerated dose had not been reached;however, the length of infusion time at the highest dose was notconsidered feasible for outpatient therapy. The ORR in 15 patients was13% (Table 1)(6). TABLE 1 Rituximab ®: Summary of Efficacy ResultsMedian Median DR TIP Study Description Indication N* ORR CR PR (months)(months) References Phase I/II, Single-Dose Relapsed B-Cell Lymphoma 15 2 (13%)  0 (0%)  2 (13%) NA{cube root} 8.1  6 Single Agent Phase I/II,Multiple-Dose Relapsed Low-, Intermediate-, 34 17 (50%)  3 (9%) 14 (41%)8.6 10.2  7 Dose-Ranging and High-Grade Lymphotna Phase II;Multiple-Dose Newly Diagnosed and Relapsed 38 38 (100%) 22 (58%) 16(42%) 35.3+ 36.7+ 21, 22 Combined with CHOP Low-Grade or FollicularB-Cell Lymphoma Phase III, Multiple-Dose Relapsed Low-Grade or 151 76(50%)  9 (6%) 67 (44%) 11.6 13.2 8, 9 Single-Agent Follicular B-CellLymphoma Phase II, Multiple-Dose Relapsed Low-Grade or 35 21 (60%)  5(14%) 16 (46%) 13.4+ 19.4+ 13 Single-Agent Follicular B-Cell LymphomaPhase II, Multiple-Dose, Relapsed Low-Grade or 38 17 (45%)  4 (11%) 13(34%) 22.3+ 25.2+ 29 Combined with Interferon Follicular B-Cell LymphomaPhase II, Multiple-Dose, Relapsed Low-Grade or 28 12 (43%)  1 (4%) 11(39%) 5.9 8.1 14 Single-Agent Follicular B-Cell Lymphoma, Bulky DiseasePhase II, Multiple-Dose, Relapsed Low-Grade or 57 23 (40%) 6 (11%) 17(29%) 15.0+ 16.7+ 19, 20 Single-Agent Follicular B-Cell Lymphoma,Retreatment Phase II, Multiple-Dose Previously Untreated 30 29 (96%) 19(63%) 10 (33%) 11+ 17+ 34 Combined with CHOP Intermediate- or High-GradeModality Lymphoma Phase II, Alternative Multiple Intermediate- orHigh-Grade B- 54 17 (32%) 5 (9%) 12 (22%) NA† 8.2+ 33 Dosing CellLymphoma

[0049] In Phase I of a Phase I/II dose-ranging study, patients received125-375 mg/m² administered as four weekly infusions. No dose-relatedtoxicities were demonstrated, and 375 mg/m² was chosen as the Phase IIdose. Tumor regressions were observed in 17 of 37 (46%) patients whoreceived this dose, including 3 (8%) complete responses (CR) and 14(38%) partial responses PR (7).

[0050] A subsequent single-arm pivotal study of Rituximab® infused at375 mg/m² weekly times four was conducted in 166 patients with relapsedor refractory, low-grade or follicular NHL (International WorkingFormulation [IWF] Types A-D and REAL classification, small lymphocyticlymphoma, Follicular center, follicular Grades I, II, III (8)). Patientswith tumor masses >10 cm or with >5000 lymphocytes/μL in the peripheralblood were excluded from this study. The median age was 58 years (105men and 61 women) and the median number of prior treatments was three.Bone marrow involvement was present in 56% of 149 patients evaluated.Forty-five percent had ≧2 extranodal sites and 41% had bulky disease (≧5cm).

[0051] Complete response required the regression of all lymph nodes to<1×1 cm² demonstrated on two occasions at least 28 days apart on neckchest, abdomen, and pelvic CT scans, resolution of all symptoms andsigns of lymphoma, and normalization of bone marrow, liver, and spleen.Partial response required a ≧50% decrease in the sum of the products ofperpendicular measurements of lesions without any evidence ofprogressive disease for at least 28 days. Patients who did not achieve aCR or PR were considered non-responders, even if a net decrease (>50%)of measurable disease was observed. Time to progression was measuredfrom the first infusion until progression.

[0052] The overall response rate (ORR) was 48% with a 6% CR and a 42% PRrate (8). The median time to progression (TTP) for responders was 13.2months and the median duration of response (DR) was 11.6 months.Twenty-two of 80 (28%) responders remain in ongoing remission at 20.9+to 32.9+ months (9).

[0053] Administration of Rituximab® resulted in a rapid and sustaineddepletion of B-cells. Circulating B-cells were depleted within the firstthree doses with sustained depletion for up to six to nine monthspost-treatment in 83% of patients. Median B-cell levels returned tonormal by 12 months following treatment. Although median NK cell countsremained unchanged, a positive correlation was observed between higherabsolute NK cell counts at baseline and response to Rituximab® (10).

[0054] Several baseline prognostic factors were analyzed to determinetheir correlation to response. Significantly, in 23 patients relapsedafter ABMT or PBSC, the ORR was 78% versus 43% in patients who did notundergo prior high-dose therapy (p<0.01). In a multivariate analysis,the ORR was higher in patients with follicular NHL as compared withsmall lymphocytic lymphoma (58% vs. 12%, p<0.01), and higher in patientswith chemosensitive relapse as compared with chemoresistant relapse (53%vs. 36%, p=0.06). No effect on response rate was associated with:age >60 years, extranodal disease, prior anthracycline therapy, or bonemarrow involvement.

[0055] A statistically significant correlation was found between themedian serum antibody concentration and response at multiple time pointsduring treatment and follow up (11).

[0056] Serum levels of antibody were higher in patients with follicularNHL compared with small lymphocytic lymphoma. Mean serum antibody wasalso inversely correlated with measurements of tumor bulk and with thenumber of circulating B-cells at baseline. The association of lowerserum antibody concentrations with higher numbers of circulating NHLcells and with higher tumor bulk suggest that the main mode of antibodyclearance is to tumor cells. The association of high serum antibodyconcentrations with response and lower tumor bulk or circulating cellssuggests that higher or more doses of Rituximab® may be necessary toinduce responses in some subsets of patients, such as those with bulkydisease.

[0057] Nevertheless, responses were seen with Rituximab® in 43% ofpatients with tumors >5 cm and in 35% of patients with tumors >7 cm,suggesting that treatment of patients with bulky disease with Rituximab®is feasible. This is surprising considering it was long thought thatantibody therapy is not conducive to treating bulky disease due to thecompact nature of the tumors.

[0058] In a study conducted in Japan (12), patients with relapsed B-celllymphoma were treated with either 250 mg/m² (N=4) or 375 mg/m² (N=8) ofRituximab® weekly times four. Of 11 evaluable patients, 8 had follicularNHL, 2 had diffuse large-cell NHL, and one had mantle-cell lymphoma. Twoof the 11 had a CR and 5 had a PR for an ORR of 64%; all responders hadfollicular histology.

[0059] Because Rituximab® serum levels and response were positivelycorrelated in previous studies, a Phase II study of eight weekly dosesof 375 mg/m² Rituximab®t was conducted in low-grade or follicular NHLpatients. The ORR was 60% in evaluable patients, with a 14% CR and a 46%PR rate. Median values for TTP in responders and DR were 13.4+ monthsand 19.4+ months, respectively (13). Though it is difficult to compareacross studies, it appears that TTP and DR may be improved by using moredoses.

[0060] Contrary to early assumptions about antibody therapy being usefulonly in micrometastatic disease, Rituximab® is quite active in high bulkdisease. In a separate study, 31 patients with relapsed or refractory,bulky low-grade NHL (single lesion of >10 cm in diameter) received 375mg/m² Rituximab® as four weekly infusions. Twelve of 28 evaluablepatients (43%) demonstrated a CR (1, 4%) or PR (11, 39%) (14).

[0061] Waldenstrom's Macroglobulinemia

[0062] Waldenstrom's Macroglobulinemia (WM) is a malignancy wherein Blymphocytes secrete excessive amounts of IgM antibodies. WM usuallyoccurs in people over sixty, but has been detected in adults in theirearly thirties. WM today is considered a rare incurable indolentmalignancy, which has in the past been treated by plasmaphoresis toreduce serum viscosity. Chemotherapeutic drugs such as an alkylatingagent and a corticosteroid are often prescribed. The most recommendeddrug for WM has been Leustatin (2CdA).

[0063] A report on seven patients with Waldenstrom's macroglobulinemiawhere the patients were treated with Rituximab® (375 mg/m² weekly times4)(15) noted responses in 4 (57%) of patients. Median progression-freesurvival was 8 months (range 3-27+months). Thus, Rituximab® should beuseful in combined therapeutic protocols, particularly withchemotherapeutic reagents such as 2CdA.

[0064] Chronic Lymphocytic Leukemia (CLL)

[0065] CLL is the liquid (leukemic) equivalent of small lymphocyticlymphoma (SLL). Patients with SLL had lower serum levels and a lowerresponse rate when treated with the standard dose of Rituximab® thanpatients with other low-grade NHL subtypes. This is probably due to thevery high levels of circulating tumor cells in patients with CLL, andbecause malignant cells involved in CLL are thought to have reducedlevels of expression of CD20 on the cell surface.

[0066] Nevertheless, the present inventors have discovered thathematologic malignancies such as CLL may be treated with Rituximab®. Arecent clinical study evaluated treatment of CLL patients at higherdoses of Rituximab® (16). All patients receive a first dose of 375 mg/m³to minimize infusion-relapsed side effects. Subsequent weekly dosages(3) remained the same but were given at an increased dose level. Sixteenpatients have been treated at dosages of 500-1500 mg/m³. Medium age was66 years (range, 25-78). Eighty-one percent had end-stage III-IVdisease. Medium white blood cell count was 40×10⁹/L (range, 4-200), Hgb11.6 g/dl (range, 7.7-14.7), platelets 75×10⁹/L (range, 16-160), medianβ₂ immunoglobulin was 4.5 mg/L (range, 3.1-9.2). Median numbers of priortherapies was 2.5 (range 1-9). Sixty percent of patients were refractoryto treatment. Two patients developed severe hypertension with the firstdose (375 mg/m³); another one received further therapy. Toxicity atsubsequent escalated dosages has been mild although no patient at the1500 mg/m³ dose level has been fully evaluated. Eight patients havecompleted therapy (4 at 500 mg/m³, 3 at 650 mg/m³, 1 at 825 mg/m³). Onepatient treated at 560 mg/m³ achieved full remission. One patient hasprogressive lympocytosis on treatment and all other patients hadreduction in peripheral blood lymphocytosis but less effect on lymphnodes. Dose escalation studies are ongoing.

[0067] Another approach to improving response in CLL patients is toupregulate the CD20 antigen using cytokines. In an in vitro study,mononuclear cells from CLL patients were incubated for 24 hours withvarious cytokines. Flow cytometry results showed significantup-regulation by IL-4, GM-CSF, and TNF-alpha (17). In fact, recent datasuggests that the upregulation of CD20 observed on CLL cells may belimited to tumor cells (Venogopal et al. Poster—PanPacific Lymphomameeting, June 1999. Cytokine-induced upregulation of CD20 antigenexpression in chronic lymphocytic leukemia (CLL) cells may be limited totumor cells). Preliminary data also suggest that interferon alpha alsoupregulates CD20 on CLL cells after only 24 hours when applied at aconcentration of 500 to 1000 U/ml.

[0068] Thus, by administering certain cytokines to CLL patients prior toor concurrently with administration of Rituximab®, the expression ofCD20 on the surface of malignant B-cells may be upregulated, therebyrendering CD20, as well as other cell surface markers such as CD 19, amore attractive target for immunotherapy. A collaborative study has beeninitiated to test for optimal cytokine doses for CD20 upregulation invivo. The study protocol involves treating ten patients initially withGM-CSF at 250 mcg/m² SQ QD×3, ten patients with IL-4 mcg/kg SQ QD×3, andten patients with G-CSF at 5 mcg/kg SQ QD×3. Mononuclear cells will beseparated by Ficon Hypaque centrifugation for apoptotic studies todetermine if upregulation of CD20 translates to enhanced killing oftumor cells by Rituximab®.

[0069] Antibody treatment of CLL can be combined with other conventionalchemotherapeutic treatments known to be useful for the treatment of CLL.The most frequently used single agent for CLL is chlorambucil(leukeran), given either as 0.1 mg/kg daily or 0.4 to 1.0 mg/kg every 4weeks. Chlorambucil is often combined with oral prednisone (30 to 100mg/m²/d), which is useful in the management of autoimmune cytopenias.Cyclophosphamide is an alternative to chlorambucil, the usual dose being1-2 g/m² every 3-4 weeks together with vincristine and steroids (e.g.,COP regimen).

[0070] Various drug combinations have been used for CLL, including COP(cyclophosphamide, Oncovin, and prednisone), and CHOP (these three drugsplus doxorubicin). Fludarabine has shown an effect in the treatment ofCLL, and gave an ORR of 50% in a group of patients treated with 25-30mg/m²/d every 3-4 weeks. http://www.cancernetwork.com. Although somepatients have been shown to be refractory for fludarabine. Such patientsmay also be resistant to 2-CdA because often, patients who arerefractory to fludarabine are also refractory to 2-CDA (O'Brien et al.N. Engl. J. Med. 330: 319-322 (1994)).

[0071] Hence, anti-CD20 antibody therapy will be particularly useful forpatients who are refractory or who have relapsed after treatment withchemotherapeutic drugs. Rituximab® therapy may also be combined withradiotherapy in these patients. TBI with a low fraction size of 15 cGyto total doses of 75 to 150 cGy has been shown to be effective in aboutone-third of patients.

[0072] A Phase II trial is currently being conducted by CALGB in CLLpatients. Rituximab® and fludarabine are administered concurrently,followed by Rituximab®D consolidation versus fludarabine inductionfollowed by Rituximab®.

[0073] Rituximab® with Myeloablative Therapy

[0074] Myeloablative therapy has yielded responses in indolentlymphomas; however, residual tumor cells may remain despite high-dosetherapy and the PBSC reinfused may contain tumor cells. Rituximab® isbeing used before stem cell mobilization and after transplant to reduceresidual CD20+ tumor cells and contamination of the bone marrow or stemcells harvested. Interim results demonstrated that no CD20+ cells weredetectable in harvested cells. Eighteen of 24 patients achievedengraftment and the treatment was well tolerated. PCR testing is ongoingto evaluate residual tumor cells (18).

[0075] Retreatment of Relapsed Low-Grade NHL with Rituximab®

[0076] A trial evaluating retreatment of 53 patients who had respondedto Rituximab® and later relapsed has been reported (19). Seven of 56evaluable patients (13%) obtained a CR and 16 a PR (29%), for an ORR of42%. Four patients who had a second response received a third treatment;3 of these responded.

[0077] After treatment with two courses of Rituximab®, one patient'stumor, initially classified as follicular, small cleaved cell NHL, nolonger expressed the CD20 antigen and was unresponsive to Rituximab® atthe time of transformation to diffuse, large-cell NHL (20).

[0078] Thus, while retreatment with Rituximab® is effective for treatingpatients who have relapsed after prior treatment with Rituximab®, theremay be an increased incidence of CD20-tumor cells after secondarytreatment. This observation supports the utility of the combinedtherapeutic treatment regimens described herein.

[0079] Combination of Rituximab® and CHOP Chemotherapy for Low-Grade NHL

[0080] Chemotherapy with cyclophosphamide, doxorubicin, vincristine, andprednisone (CHOP) is an effective first-line therapy for low-grade orfollicular NHL. Though initial response rates are high, relapseeventually occurs and subsequent chemotherapy regimens produceremissions with shorter durations. A Phase II trial was initiated toevaluate the combination of CHOP and Rituximab® (21) in newly diagnosedand relapsed low-grade or follicular NHL because their mechanisms ofaction are not cross-resistant, and Rituximab® is synergistic withcertain cytotoxic drugs, including doxorubicin (5).

[0081] Twenty-nine of 38 patients received no prior anticancer therapy.CHOP was administered at standard doses every three weeks for six cycleswith six infusions of Rituximab® (375 mg/m²). Rituximab® infusions 1 and2 were administered on Days 1 and 6 before the first CHOP cycle, whichstarted on Day 8. Rituximab® infusions 3 and 4 were given 2 days beforethe third and fifth CHOP cycles, respectively, and infusions 5 and 6were given on Days 134 and 141, respectively, after the sixth CHOPcycle.

[0082] In this combination study, 100% of the 38 patients treatedresponded (CR, 58%; PR, 42%). Of 35 evaluable patients who completedtreatment, there were 63% CR, and 37% PR (21). Median DR is 35.3+ monthswith median progression-free survival not reached after a medianobservation time of 36.7+ months. Twenty patients are still in remissionafter 36+ months to 53.4+ months (22). This DR is impressive even forfirst-line treatment, and 24% of this trial population had relapsedafter chemotherapy.

[0083] In a study to be conducted by CALGB, 40 patients with low-gradeNHL will receive Rituximab® weekly times 8 and oral cyclophosphamidedaily starting on Day 8. Twenty patients will receive Rituximab® alonefor 8 weekly doses.

[0084] A Phase III study conducted by ECOG in patients with low-gradeNHL is comparing the combination of cyclophosphamide and fludarabine(Ann A) with standard CVP therapy (Arm B). In the randomization to Arm Aor Arm B, patients are stratified by age, tumor burden, histology, and Bsymptoms. Responders in both arms will undergo a second randomization toRituximab® maintenance therapy (375 mg/m² weekly times 4 every 6 monthsfor 2 years (Arm C) or to observation (Arm D).

[0085] Combination of Rituximab® with Cytokines

[0086] Rituximab® Plus Interferon Alpha

[0087] Interferon is a cytokine involved in modulating the immune system(23). Mechanisms by which interferon may increase the effectiveness ofantibodies include the potentiation of antigen expression (24),increased targeting of antibodies into tumors (25,26), and enhancedcytotoxicity of immunotoxins (27).

[0088] In a combination trial, interferon-alpha (Roferon-A), a cytokinewith a single-agent clinical activity in NHL (28), and Rituximab® weregiven to patients with relapsed low-grade or follicular NHL.Interferon-alpha (2.5 or 5 MIU) was administered subcutaneously, threetimes weekly for 12 weeks. Rituximab® was administered by IV infusionweekly for four doses (375 mg/m²) starting on the fifth week oftreatment. The ORR was 45% (17/38 patients); 11% had a CR and 34% had aPR. Kaplan-Meier estimates of the median DR and TTP in responders were22.3+ and 25.2+ months, respectively (29). Previous combination studiesof interferon-alpha and chemotherapeutic regimens containinganthracyclines yielded prolonged time to progression, but did notconsistently increase response or survival rates (30-32). These earlyresults suggest that the combination of Rituximab® and interferon-alphamay prolong the time to progression relative to Rituximab® alone.

[0089] Rituximab® Plus G-CSF

[0090] In a separate study, Rituximab® and G-CSF are being evaluated inrelapsed low-grade NHL. It has been demonstrated in vitro as well as invivo in healthy volunteers that G-CSF, via its effect on myeloidprecursor cells, induces FcRI-positive neutrophils that are capable offunctioning as effector cells in ADCC. Therefor, a Phase I/II study wasinitiated to evaluate the toxicity and efficacy of the combinedtreatment.

[0091] Both in Phase I and Phase II, patients were administered astandard dose of G-CSF (5 μg/kg/day) administered for three days,starting 2 days before administration of Rituximab®. Phase I consistedof a dose escalation of Rituximab® (125, 250, or 375 mg/m² weekly×4).Early results in 9 patients evaluated so far yielded an ORR of 67% (44%CR, 22% PR) with minor toxicity in 8 of the 9 patients (33). The mostfrequent adverse events were fever (4/8 patients), rhinitis (4/8),chills (3/8) and headaches (3/8), which were comparable to the adverseevents observed previously in administration of Rituximab® alone. ThePhase II part of the study has been initiated, which will examine theefficacy of the combination of G-CSF and 375 mg/m² Rituximab®×4.

[0092] Rituximab® Plus IL-2

[0093] High-dose therapy with autologous peripheral blood stem cells(PBSC) or bone marrow (BM) rescue has been used to treat NHL, howeversuccess remains limited by the high risk of relapse, which is 50-80%. Inan effort to improve durable remissions post-transplant, immunotherapyincluding high dose and low dose therapy with IL-2 has been studied in anumber of treatment centers. Such studies have suggested that IL-2therapy does demonstrate early post-transplant anti-Tumor activity.

[0094] Initially following autologous transplant, patients displaydelayed immune reconstitution which potentially results in diminishedimmune-mediated tumor eradication (43, 44). Indeed, it has been shownthat both CD$+ T cells and cytotoxic CD8+ T cells are depressed (45-49).In vitro assays have demonstrated a profound suppression of T cellcytolytic and proliferative responses as well as decreased production ofIL-2 in response to mitogens and soluble antigens. However, soluble IL-2is able to restore these immune responses suggesting that immune cellsin patients after autologous transplant are capable of responding toexogenous L-2 (47). Peripheral blood NK activity also remains lowerfollowing BMT than control values and the NK activity is also augmentedby addition of exogenous IL-2 (49). These data suggest thatadministration of IL-2 to patients shortly after stem cell transplantmay enhance immune responsiveness at a critical period when tumor burdenis minimal and when immune responsiveness in the absence of IL-2 islacking.

[0095] For instance, Caligiuru et al. have shown that IL-2(Hoffman-LaRoche) administered at 0.45×10⁶ U/M²/day by 24 hour CIV for12 weeks was able to expand the absolute number of CD56 bright NK cells(50-52). This regimen was administered to non-transplant patients in theoutpatient setting with little toxicity.

[0096] Animal models have shown that non-LAK inducing low doses of 1L-2dramatically enhances anti-tumor activity when administered withtumor-specific T effector cells (53). In addition, Soiffer et al. (54)administered low doses of IL-2 to 13 autologous BMT or T cell depletedallogeneic BMT recipients undergoing treatment for relapsed leukemia orlymphoma. Enhanced immunological responsiveness was demonstrated in thelaboratory with a 5- to 40-fold increase in circulating CD56 brightCD16+ CD3− NK cells. Moreover, this low dose regimen of IL-2 resulted inaugmented in vitro killing of the NK targets K562. When Soiffer et al.(55) updated the outcome of 29 allogeneic BMT patients who received lowdose IL-2, they found superior survival for these patients (70%)compared to histological controls (30%, p=0.41).

[0097] Lauria et al. (56) treated 11 patients with high grade NHL at amedian of 42 days after ABMT with IL-2 at a dose of 2×10⁶ IU/m² qod fortwo weeks and then 3×10⁶ IU/m² twice a week for a year. Phenotypicanalysis showed a persistent and significant (p=0.001) increase in theproportion and absolute number of total lymphocytes and especially ofboth CD 16 and CD56 NK cells after 6 months of therapy. None of thepatients progressed with a median follow-up of twenty-two months (range10-42 months) after starting therapy. In addition, two patients withresidual disease after ABMT, one in the liver and second in the lymphnodes, obtained a complete response after 7 and 10 months of IL-2therapy.

[0098] Vey et al. (57) treated 25 patients with refractory or relapsedHD (11 patients) and NHL (14 patients) with low dose IL-2. 48% of thepatients had resistant disease at transplant and 84% achieved CR afterABMT. IL-2 was started at a mean of 54 days after transplant andconsisted of a first cycle of 5 days followed by 4 cycles of 2 daysevery other week. Patients received a mean of 160×10⁶ IU/m2 of IL-2.After a five year follow-up, the probability of survival and DFS is 72%(HD 73% and NHL 70%) and 45% (HD 36% and NHL 48%).

[0099] A group at the Fred Hutchinson Cancer Research Center (FHCRC) hasrecently found that low dose IL-2 therapy was well-tolerated in theoutpatient setting, and that remissions in patients treated with lowdose IL-2 tended to be longer than without IL-2 treatment. IL-2 therapywas associated with an increase in the number of certain populations ofimmune cells, including CD8+ CD69+ cells; CD16+ CD8+ cells; CD16+ CD69+cells; CD16+ CD56+ cells; CD16+ CD122+ cells; CD16+ Dr+ cells; and CD8+CD56+ cells. There was also an increase in the expression of lyticactivity against the timor targets K562 and Daudi, with a median of5.9-fold and 6.5-fold increase, respectively. Relapses, when theyoccurred, occurred at a median of 17.8 months after transplant, andtherefor remissions were reported to be characteristically longer thanwhat was historically seen in transplant recipients without IL-2therapy.

[0100] Given the encouraging data gathered from single therapy studieswith IL-2 on ABMT transplant recipients, it seemed reasonable to combineIL-2 therapy with Rituximab® post transplant, given that Rituximab'sbiological activity appears to be mediated through ADCC andcomplement-mediated lytic activity. Thus, a Phase I trial has beeninitiated in collaboration with the FHCRC to evaluate the safety andpotential efficacy of a combined therapeutic regimen.

[0101] A separate Phase II study is also being performed to evaluate theefficacy and the incidence of HACA formation in patients receivinglow-dose IL-2 and Rituxan®. A specific objective of this study is toassess whether ADCC is enhanced by in vivo exposure to IL-2 and whetherADCC activity correlates with clinical response. Inclusion criteria forpatients are histologically confirmed stage 1I-IV low-grade, follicularB-cell or mantle cell ylmphoma. Mantle cell lymphoma, for the purposesof this clinical study, is defined as CD5+, CD23− (if available) and/orbcl-1+ by immunohistochemistry. Patients who did not respond to or haverelapsed following their first treatment with a standard therapy, i.e.,chemotherapy, radiotherapy, ABMT and/or immunotherapy, are eligible.

[0102] Rituximab® Plus GM-CSF for the Treatment of Relapsed Low Grade orFollicular B-Cell Lymphoma

[0103] Two separate Phase II trials have also been initiated to test theefficacy of combined treatment with Rituximab® and GM-CSF. One studyinvolves 40 patients with relapsed low grade B-cell lymphoma, andcomprises administering Rituximab® at 375 mg/m² weekly×4 (d. 1, 8, 15,22) and GM-CSF (Leukine, Immunex) at 250 mcg sc three times weekly for 8weeks, starting one hour before the first dose of Rituximab®. This studywill be used to evaluate the clinical efficacy (overall response rate(ORR), overall complete response rate, time to progression andfailure-free survival) of the combined therapeutic regimen, tocharacterize the safety (qualitative, quantitative, duration andreversibility of adverse events) of the combined therapy, and todetermine the effects of the combined therapy on relevant lymphocytesubsets and cytokines. The second study plans to also monitorimmunologic parameters to assess the mechanism of killing (complement C3and C4, CH50, flow cytometry for CD3, CD4, CD8, CD 16, CD19 and CD56 andADCC assay).

[0104] Rituximab® Plus Gamma-Interferon

[0105] Gamma-interferon may also be useful in combined therapy withRituximab® for treating patients with low-grade or higher-gradelymphomas. It is has recently been found that gamma-interferonupregulates CD20 expression on multiple myeloma (MM) patient plasmacells, patient B-cells, as well as on normal donor B-cells (Treon etal., Lugano, 1999). In fact, Treon and colleagues have shown thatgamma-interferon augments binding of these cells to Rituximab®.Induction of CD20 expression on plasma cells occurred in a dosedependent manner, with upregulation seen with as little as 1 U/ml ofinterferon gamma. A plateau occurred at 100 U/ml at 48 hours. Thus,gamma-interferon may also be beneficial when administered in combinationwith Rituximab®.

[0106] Intermediate-Grade and High-Grade NHL

[0107] Single-Agent Studies

[0108] In a study conducted in Europe and Australia, alternative dosingschedules were evaluated in 54 relapsed or refractory intermediate- orhigh-grade NHL patients (34). Rituximab® was infused at 375 mg/m² weeklyfor 8 doses or at 375 mg/m2 once followed by 500 mg/m² weekly for 7doses. The ORR was 31%; (CR 9%, PR 22%) no significant differencebetween the dosing regimens was observed. Patients with diffuselarge-cell lymphoma (N=30) had an ORR of 37% and those with mantle-celllymphoma (N=12) had an ORR of 33%.

[0109] Combination of Rituximab® and CHOP Chemotherapy

[0110] In another study, 31 patients with intermediate- or high-gradeNHL (19 females, 12 males, median age 49) received Rituximab® on Day 1of each of six 21-day cycles of CHOP (35). Of 30 evaluable patients,there were 19 CR (63%) and 10 PR (33%), for an ORR of 96%. This regimenwas considered well tolerated and may result in higher response ratesthan with Rituximab® or CHOP alone.

[0111] The NCI Division of Cancer Treatment and Diagnosis iscollaborating with IDEC Pharmaceuticals Corporation to exploreRituximab® treatment in other indications. A Phase II trial of CHOPversus CHOP and Rituximab® is being conducted by ECOG, CALGB, and SWOGin older patients (>60 years) with mixed, diffuse large cell, andimmunoblastic large cell histology NHL (N=630 planned). This studyincludes a secondary randomization to maintenance with Rituximab® versusnon-maintenance.

[0112] A Phase III trial of Rituximab® and CHOP in 40 patients withpreviously untreated mantle-cell lymphoma is also ongoing at the DanaFarber Institute. Rituximab® is administered on Day 1 and CHOP is givenon Days 1-3 every 21 days for 6 cycles. Accrual for this study has beencompleted. A Phase II trial of CHOP followed by Rituximab® in newlydiagnosed follicular lymphoma conducted by SWOG has also been completed.Results of these two trials are being analyzed.

[0113] A Phase II trial of CHOP and Rituximab® versus CHOP alone inHIV-related NHL conducted by the AIDS Malignancy Consortium is ongoing;120 patients are planned.

[0114] Rituximab® after Myeloablative Therapy Relapse

[0115] Rituximab® has shown promising early results in patients withrelapsed intermediate-grade NHL after high-dose therapy with autologousPBSC support. Six of seven patients responded (1 CR and 5 PR) and onepatient had stable disease; therapy was well tolerated (36).

[0116] Safety Experience

[0117] Adverse events and clinical laboratory data from 315 patients inthe five single-agent U.S. studies were combined to provide a safetyprofile of Rituximab® in patients with low-grade or follicular NHL. Themajority of adverse events were infusion-related and occurred withdecreasing frequency after the first infusion. The most commoninfusion-related events were fever (49%), chills (32%), nausea (18%),fatigue (16%), headache (14%), angioedema (13%), pruritus (10%), andoccasionally, hypotension (10%) and bronchospasm (8%). During thetreatment period (up to 30 days following the last dose), 10% ofpatients experienced Grade 3 or 4 adverse events, which were primarilyinfusion-related or hematologic. Thrombocytopenia (<50,000platelets/mm³) occurred in 1.3% of patients, neutropenia (<1000/mm³)occurred in 1.9%, and anemia (<8 gm/dL) occurred in 1.0%. AlthoughRituximab® induced B-cell depletion in 70%-80% of patients, abnormallydecreased serum immunoglobulins were observed in a minority of patientsand the incidence of infection did not appear to be increased.

[0118] Hypotension requiring interruption of the Rituximab® infusionoccurred in 10% of patients and was Grade 3 or 4 in 1%. Angioedema wasreported in 13% of patients and was considered serious in one patient.Bronchospasm occurred in 8% of patients; 2% were treated withbronchodilators. A single report of bronchiolitis obliterans was noted.Most patients experienced no further infusion-related toxicities by thesecond and subsequent infusions. The percentage of patients reportingadverse events upon retreatment was similar to that reported followingthe first course (14).

[0119] Four patients developed arrhythmias during Rituximab® infusion.One of the four discontinued treatment because of ventriculartachycardia and supraventricular tachycardias. The other three patientsexperienced trigeminy (N=1) and irregular pulse (N=2) and did notrequire discontinuation of therapy. Angina was reported during infusionand myocardial infarction occurred four days post-infusion in onesubject with a prior history of myocardial infarction.

[0120] The overall incidence of adverse events and Grade 3 and 4 adverseevents was higher in patients with bulky disease than in patients withnon-bulky disease. The incidence of dizziness, neutropenia,thrombocytopenia, myalgia, anemia, and chest pain was higher in patientswith lesions >10 cm. The incidence of Grade 3 or 4 neutropenia, anemia,hypotension, and dyspnea was also higher in patients with bulky diseasecompared with patients with lesions <10 cm (19).

[0121] Since FDA approval of Rituximab® for treatment of relapsed orrefractory low-grade or follicular NHL in 1997, an estimated 17,000patients have been treated. In May, 1998, descriptions of eightpost-marketing reports of severe infusion-related adverse eventsassociated with the use of Rituximab® that resulted in fatal outcomeswere summarized. In seven of the eight fatalities, severe symptomsoccurred during the first Rituximab® infusion. The cause of death wasnot reported or remains unknown for two of the eight cases. Severerespiratory events, including hypoxia, pulmonary infiltrates, or adultrespiratory distress syndrome contributed to six of the eight reporteddeaths. One patient had a pretreatment lymphocyte count of 600,000/mm³;another, a creatinine of 8; a third, a respiratory rate of 40; and afourth, pancytopenia. Patients with a high tumor burden or with a highnumber of circulating malignant cells may be at higher risk and thesepatients should be monitored closely throughout each infusion.

[0122] Most of the adverse events recently described were previouslyobserved in Rituximab® clinical studies. One notable exception is aninfusion-related syndrome associated with rapid tumor lysis, that wasreported in six patients with high numbers of circulating tumor cells(37,38). This syndrome was characterized by fever, rigors, bronchospasmwith associated hypoxemia, a rapid decline in peripheral lymphocytes,laboratory evidence of tumor destruction, and transient, severethrombocytopenia. These patients had diagnoses of B-prolymphocyticleukemia (N=2), chronic lymphocytic leukemia (N=2), mantle-cell lymphoma(N=1), or transformed NHL (N=1); all had elevated circulatinglymphocytes, bulky adenopathy, and organomegaly. Although five of thesesix patients required hospitalization, symptoms resolved and subsequentRituximab® treatments were well tolerated; the last patient refusedfurther therapy and died of progressive disease two weeks later.

[0123] In a separate report of seven patients with CLL and one patientwith mantle-cell lymphoma, tumor lysis syndrome was observed after thefirst Rituximab® infusion in those patients with lymphocyte counts>10×10⁹L (39).

[0124] Radioimmunotherapy with ⁹⁰Yttrium-Labeled Anti-CD20 Antibody inCombination with Rituximab®

[0125] Another therapeutic approach to NHL under evaluation is aradiolabeled anti-CD20 antibody (IDEC-Y2B8) in combination withRituximab®. IDEC-Y2B8 (⁹⁰Y-ibritumomab tiuxetan) is a murine IgG₁ kappaanti-CD20 antibody conjugated to ⁹⁰Y via a chelator, MX-DTPA, which iscovalently bound to the antibody. Rituximab® (250 mg/m2) is administeredprior to IDEC-Y2B8 to deplete peripheral B lymphocytes and improvebiodistribution of the radiolabeled antibody.

[0126] In a recently reported Phase I/II study (40-42), patients withlow-grade NHL (N=34), intermediate-grade NHL (N=14), or mantle-celllymphoma (N=3) were treated with IDEC-Y2B8. The median age was 60, 71%were male, and 96% were Caucasian. Of 51 patients with relapsed orrefractory NHL, 34 (67%) responded to single doses of 0.2, 0.3, or 0.4mCi/kg of IDEC-Y2B8. The ORR was 82% (28/34) for patients with low-gradeor follicular NHL and was 43% (6/14) for patients withintermediate-grade lymphoma. No patients with mantle-cell diseaseresponded.

[0127] A Phase III randomized study comparing IDEC-Y2B8 with Rituximab®(375 mg/m² weekly times 4) for treatment of low-grade follicular ortransformed NHL patients is ongoing. Another Phase III trial is alsobeing conducted in patients with relapsed NHL who are refractory toRituximab®.

SUMMARY

[0128] In the absence of curative therapy for NHL, the objective oftreatment is to achieve control of the disease for a meaningful durationand provide relief of tumor-related symptoms without undue toxicity.Treatment with Rituximab® is a brief, 22-day outpatient therapy withlimited adverse events in most patients. In clinical studies, 50% ofevaluable relapsed or chemotherapy refractory low-grade or follicularNHL patients achieved complete or partial responses. These responseswere durable without maintenance therapy; the median TTP for responderswas 13.2 months and the median DR was 11.6 months in the pivotal study.

[0129] Rituximab® is approved as a safe and effective treatment forpatients with relapsed low-grade or follicular B-cell NHL. It hassignificant clinical activity, a novel mechanism of action, and comparesfavorably with alternative therapies in response rate and responseduration. Completion of ongoing studies will verify the role ofalternative Rituximab® regimens and Rituximab® in the treatment of otherCD20+ B-lymphocyte malignancies.

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What is claimed:
 1. A method for treating relapsed B-cell lymphomacomprising administering to a patient having relapsed the B-celllymphoma a therapeutically effective amount of an anti-CD20 antibody. 2.The method of claim 1, wherein said patient was treated previously withan anti-CD20 antibody.
 3. The method of claim 1, wherein said patientpreviously underwent a bone marrow or stem cell transplantation.
 4. Themethod of claim 1, wherein said patient previously underwentradiotherapy.
 5. The method of claim 1, wherein said patient previouslyunderwent chemotherapy for said B-cell lymphoma.
 6. The method of claim5, wherein said chemotherapy is selected from the group consisting ofCHOP, ICE, Mitozantrone, Cytarabine, DVP, ATRA, Idarubicin, hoelzerchemotherapy regime, La La chemotherapy regime, ABVD, CEOP, 2-CdA, FLAG& IDA with or without subsequent G-CSF treatment), VAD, M & P, C-Weekly,ABCM, MOPP and DHAP.
 7. A method for treating a subject having B-celllymphoma, which subject has not exhibited appreciable tumor remission orregression after administration of a chimeric anti-CD20 antibody,comprising administering to said patient a radiolabeled anti-CD20antibody.
 8. The method of claim 7, wherein said radiolabeled anti-CD20antibody is administered from about one week to about two years aftersaid administration of said chimeric anti-CD20 antibody.
 9. The methodof claim 8, wherein said radiolabeled anti-CD20 antibody is administeredfrom about one week to about nine months after said administration ofsaid chimeric anti-CD20 antibody.
 10. The method of claim 1, whereinsaid anti-CD20 antibody is a chimeric anti-CD20 antibody.
 11. The methodof claim 10, wherein said chimeric antibody is C2B8 (Rituximab®).
 12. Amethod for treating B-cell lymphoma comprising administering asynergistic therapeutic combination comprising at least one anti-CD20antibody and at least one cytokine, wherein the therapeutic effect isbetter than the additive effects of either therapy administered alone.13. The method of claim 12, wherein said at least one cytokine isselected from the group consisting of alpha interferon, gammainterferon, IL-2, GM-CSF and G-CSF.
 14. The method of claim 13, whereinsaid anti-CD20 antibody and said alpha interferon, gamma interferon,IL-2, GM-CSF or G-CSF is administered sequentially, in either order, orin combination.
 15. The method of claim 12, wherein said anti-CD20antibody is a chimeric antibody.
 16. The method of claim 15, whereinsaid chimeric anti-CD20 antibody is C2B8 (Rituximab®).
 17. A method fortreating B-cell lymphoma comprising administering to a patient atherapeutically effective amount of anti-CD20 antibody before, during orsubsequent to a chemotherapeutic regimen.
 18. The method of claim 17,wherein said chemotherapy regimen is selected from the group consistingof CHOP, ICE, Mitozantrone, Cytarabine, DVP, ATRA, Idarubicin, hoelzerchemotherapy regime, La La chemotherapy regime, ABVD, CEOP, 2-CdA, FLAG& IDA with or without subsequent G-CSF treatment), VAD, M & P, C-Weekly,ABCM, MOPP and DHAP.
 19. The method of claim 17, wherein said anti-CD20antibody is a chimeric antibody.
 20. The method of claim 19, whereinsaid chimeric antibody is C2B8 (Rituximab®).
 21. A method for treatingB-cell lymphoma comprising administering to a patient a therapeuticallyeffective amount of an anti-CD20 antibody before, during or subsequentto a bone marrow or stem cell transplant.
 22. The method of claim 21,wherein said anti-CD20 antibody is a chimeric anti-CD20 antibody. 23.The method of claim 22, wherein said chimeric anti-CD20 antibody is C2B8(Rituximab®).
 24. A method of reducing residual CD20+ tumor cells inbone marrow or stem cells before or after myeloablative therapy byadministering to a patient an anti-CD20 antibody.
 25. The method ofclaim 24, wherein said anti-CD20 antibody is a chimeric anti-CD20antibody.
 26. The method of claim 25, wherein said chimeric anti-CD20antibody is C2B8 (Rituximab®).
 27. The method of claim 1, wherein saidB-cell lymphoma is selected from the group consisting of lowgrade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL)NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL,chronic lymphocytic leukemia (CLL), high grade immunoblastic NHL, highgrade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulkydisease NHL, mantle cell lymphoma, AIDS-related lymphoma andWaldenstrom's Macroglobulinemia.
 28. The method of claim 12, whereinsaid B-cell lymphoma is selected from the group consisting of lowgrade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL)NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL,chronic lymphocytic leukemia (CLL), high grade immunoblastic NHL, highgrade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulkydisease NHL, mantle cell lymphoma, AIDS-related lymphoma andWaldenstrom's Macroglobulinemia.
 29. The method of claim 17, whereinsaid B-cell lymphoma is selected from the group consisting of lowgrade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL)NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL,chronic lymphocytic leukemia (CLL), high grade immunoblastic NHL, highgrade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulkydisease NHL, mantle cell lymphoma, AIDS-related lymphoma andWaldenstrom's Macroglobulinemia.
 30. The method of claim 21, whereinsaid B-cell lymphoma is selected from the group consisting of lowgrade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL)NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL,chronic lymphocytic leukemia (CLL), high grade immunoblastic NHL, highgrade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulkydisease NHL, mantle cell lymphoma, AIDS-related lymphoma andWaldenstrom's Macroglobulinemia.
 31. The method of claim 24, whereinsaid B-cell lymphoma is selected from the group consisting of lowgrade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL)NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL,chronic lymphocytic leukemia (CLL), high grade immunoblastic NHL, highgrade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulkydisease NHL, mantle cell lymphoma, AIDS-related lymphoma andWaldenstrom's Macroglobulinemia.